The formation of background staining has limited the sensitivity, reproducibility, and quantitative analyses of most polyacrylamide silver staining methods. In the first applications of silver staining as a detection method for proteins separated on polyacrylamide gels, the problem of excessive background was addressed. A photographic "reducer" containing sodium thiosulfate was used to reduce the excessive background staining. However, this procedure not only affected the background, but also the silver densities in the stained protein bands or spots. Furthermore, the "reducer" effect on protein bands or spots is not necessarily proportional. This non-proportional loss of silver grains has limited this use of thiosulfate, particularly in quantitative applications.
It was found that background staining of the ammoniacal silver stain could also be reduced by pretreating the gels with diluted thiosulfate solutions prior to silver reduction or image development. However, such pretreatment often results in the inability to visualize trace proteins. The use of thiosulfate to eliminate background staining also results in another problem, i.e. the loss of image permanence.